Within the field of IVF (in-vitro fertilization) it is desirable to identify the number and complement of chromosomes within the cells of an embryo prior to implantation. There is increasing evidence that one of the most important factors influencing embryo viability is chromosome imbalance, including copy number gain/loss and whole chromosome aneuploidy (abnormal number of chromosomes).
Current methods for testing first involve isolation of the genetic material which is representative of the embryo for testing. Samples currently used in the analysis of aneuploidy are a polar body biopsy associated with the oocyte, a single cell from blastomere biopsy (associated with the day 3 embryo), or trophoectoderm biopsy (associated with the day 5 embryo, or blastocyst). In some cases, however, samples taken at other or multiple points in the process prove more effective. The polar body or cell(s) are then tested via a choice of methods to detect copy number imbalance. For the purposes of the present application, such testing methods will be referred to as preimplantation genetic screening (PGS), although the term PGD is often encountered in the literature. The term PGS shall also include testing of polar bodies to access oocyte quality, for example, to enable informed egg banking.
Comparative genomic hybridization (CGH) is a technique that has been employed to detect the presence and identify the location of amplified or deleted sequences in genomic DNA, corresponding to so-called changes in copy number. Typically, genomic DNA is isolated from normal reference cells, as well as from test cells. The two nucleic acid samples are differentially labeled and then hybridized in-situ to metaphase chromosomes of a reference cell. The repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means. Chromosomal regions in the test cells which are at increased or decreased copy number can be identified by detecting regions where the ratio of signal from the two DNAs is altered. The detection of such regions of copy number change can be of particular importance in the diagnosis of genetic disorders.
Metaphase CGH, as described above, has also been applied to and has the ability to screen all chromosomes for abnormalities. For CGH analysis to be applied to a PGS context, amplification of the entire genome is required to increase the quantity of DNA from a single cell (5-10 pg) to levels suitable for metaphase CGH (1 μg) prior to analysis. Commonly used methods for amplification include DOP-PCR (Telenius et al., 1992) or more recently whole-genome amplification kits such as, GENOMEPLEX (Rubicon genomics) and REPLI-G (Qiagen). The main problem with using metaphase CGH in a clinical setting is that it can take around 4 days to complete, which is not compatible with the time frame required for the pre-implantation of embryos in IVF, without the freezing of embryos and implantation occurring in the following cycle. In addition, the method is technically challenging and requires high levels of expertise to carry out and analyze. These difficulties have limited the widespread use of metaphase CGH in PGS.
Pinkel et al. in 1998 and 2003 disclosed the technique which has become widely known as array comparative genomic hybridization, hereafter referred to as arrayCGH. In 1998, Solinas-Toldo et al. described a similar “Matrix-based comparative genomic hybridization” approach.
The arrayCGH technique relies on similar assay principles to CGH with regard to exploiting the binding specificity of double stranded DNA. In arrayCGH, the metaphase chromosomes of a reference cell are replaced with a collection of potentially thousands of solid-support-hound unlabelled target nucleic acids (probes), for example, an array of clones which have been mapped to chromosomal locations. ArrayCGH is thus a class of comparative techniques for the high throughput detection of differences in copy number between two DNA samples, both of which are hybridized to the same hybridization area. It has advantages over CGH in that it allows greater resolution to be achieved and has application to the detection and diagnosis of genetic disorders induced by a change in copy number, in addition to other areas where copy number detection is important. While the particulars vary, a range of different probe types can be used, including those encountered in oligonucleotide, PAC, and bacterial artificial chromosomes (BAC) arrays.
ArrayCGH is currently being used to support the efforts of clinicians in the investigation of genomic imbalance in constitutional cytogenetics and increasingly in oncology. These applications are incredibly demanding such that the microarrays designed for these applications must be produced to far more rigorous standards than those used in academic or pre-clinical research applications.
ArrayCGH has an advantage over metaphase CGH in that the interpretation is much simpler and easily automated; in addition the time taken for the complete analysis is shorter. ArrayCGH can be used to detect aneuploidy in single cells and has been successfully applied to PGS. Single cells have to be amplified for the technique and the same methods are employed as those used in metaphase CGH. ArrayCGH allows comprehensive analysis of the whole genome to be completed within 48 hours, which allows aneuploidy screening without cryopreservation in PGS.
In order to achieve optimal assay results, arrayCGH requires the test and reference samples to be well matched in terms of quality and concentration. In the context of PGS, the starting point for any analysis is the genetic material which is as representative as possible of the fertilized embryo, or oocyte in the context of egg banking. Currently it is possible to examine the genetic material contained within a polar body or a blastomere, a single cell extracted from an 8 cell embryo, or alternatively a small number of cells from a blastocyst or associated biopsy. As only a limited amount of DNA can be obtained from such material, most downstream analyses require DNA amplification procedures to be used in order to produce large numbers of copies of the starting material. It is to be understood that polar bodies are ejected as a fertilization process begins and there are two of them, PB1 and PB2. The process is not straight forward. Herein, the term “polar body” can comprise a body ejected or biopsied from a primary or secondary oocyte.
While un-amplified genomic reference material may be used, corresponding arrayCGH results can show high noise levels due to poor matching of amplified test with un-amplified reference. Thus, reference material used in this context is often a ‘normal’ pooled DNA sample diluted to contain a broadly similar quantity of DNA as a small number of single cells. This diluted reference material is then amplified using the same method as the test sample. Even though these steps are taken to match the properties of the test and reference samples this is not always effective and the clarity of results can vary. This may be for a wide variety of reasons, including: minor errors in the quantification of the starting DNA and hence variable quantities of DNA in the diluted sample; variation due to the stochastic nature of the amplification process; amplification of impurities in the sample which are not present in the reference; low sample DNA “quality” leading to increased non-specific amplification; variability in the quantity and type of reagents used in the extraction and storage of samples. In all cases, the resultant differences between amplification of sample and reference can both alter and obscure the results of the true amplification, leading to altered arrayCGH profiles, and frequently increased noise and suppressed dynamic range.
PGS is a diagnostic application, and it is standard practice for each experiment to include an internal control to demonstrate successful functioning of the experiment, and also to assess variation in dynamic range between experiments which, for example, may arise due to the amplification issues described previously. When using arrayCGH, the most commonly used approach to address this problem is to use a reference sample with known copy number gains/losses relative to the test sample. These can then be used as a measure of performance for each individual assay.
Most frequently, the reference sample is sex-mismatched against the test, giving a shift on the log2 ratio of test over reference for the X and Y chromosomes, and consequently a measure of dynamic range. While applicable in many contexts, in the case of PGS, however, it is generally not possible to know a priori the sex of the sample, especially in aneuploidy screening of blastomere or blastocyst biopsy samples that could be either sex. The use of a single reference as internal control is therefore not reliably possible. Moreover, selection of a single appropriate reference, with a known copy number imbalance in regions other than the sex chromosomes, to a test sample is generally not possible, as the degree of copy number variation in embryos/oocytes is extremely high, and current research indicates that there are no regions which are predictably stable. In some embodiments, the selection of an embryo for implantation can be made on the basis of aneuploidy status. In other embodiments, selection is made on the basis of smaller genetic aberrations.
An alternative is to use a reference which includes non-human control sequences. However, this approach is less than ideal as it is difficult to choose non-human sequences which accurately mimic the behavior of human sequences. In any case, the use of non-human control sequences can suffer from the same amplification biases, and other biases, and as such choice of a single reference can be challenging.
To overcome this problem in PGS, it would be necessary to carry out two conventional arrayCGH hybridizations to analyze a single test sample, one against a male and another against a female reference to ensure that the assay is working correctly. However, the cost associated with this approach is unacceptably high for the application.
Where two or more cells are taken from an embryo, for example, from a blastocyst/trophoectoderm), the possibility of mosaicism in the test sample becomes significant in a PGS context, as embryos are frequently mosaic. To complicate matters, the number of cells taken From the embryo may be unknown due to inaccuracy of biopsy methods. While arrayCGH can detect mosaicism, it provides no means to directly quantify this mosaicism due to a lack of sufficiently sophisticated internal controls and furthermore, for the same reason, may mistake experimental noise for mosaicism. ArrayCGH's reliance on a single reference sample is again problematic in this context.
ArrayCGH requires contrasting fluorescent dyes to label the test and reference samples. The popular dye pair Cy3 and Cy5 is often used for arrayCGH. The Cy5 dye is susceptible to degradation by ozone in the environment and particularly when combined with high humidity, this influence on assay quality can lead to the loss of experimental data. ArrayCGH is used wherein two fluorescently labeled samples are competitively hybridized to the same hybridization area, such that through ratiometric comparison relative gain or loss of genetic material can be ascertained. Typically, one sample is a test sample of unknown genetic make-up and one sample is a reference sample known to have normal copy number, where normality is defined by the application in question. ArrayCGH is a powerful and robust technique, however the PGS application presents unique technical challenges. In some embodiments, the assessment of chromosomal content of an embryo, can be made either directly through taking cells after fertilization, or indirectly through assessing polar bodies and thus the oocyte generating the embryo. In some embodiments, an application exists whose only purpose is to assess the content of the oocyte, and no embryo is necessarily generated. This is referred to herein as egg banking.
Buffart et al. (2008) suggest a modified arrayCGH technique that they term “across arrayCGH” (aaCGH), as an improvement to the current technologies. AaCGH is similar to arrayCGH, but instead of hybridization of the test and reference sample to a single hybridization area, test and reference samples are compared from separate hybridization areas. This method, independently developed by the authors of this patent, offers advantages in cost and potentially in data quality as it removes any noise due to dye bias. The quality of the profiles obtained using aaCGH were reported to match or even surpass those obtained using regular dual channel arrayCGH. The reference is described as being hybridized at the same time, on the same slide, as the test using a multi format array, and the test and reference are labeled with the same fluorescent dye. They compare a single test sample with a single reference sample. The method does not, however, overcome the unique challenges of PGS.
SNP array techniques, as distinct from arrayCGH, may also be used to determine copy number in DNA samples, and have also been deployed for PGS applications. SNP arrays over screening of all chromosomes and allow concurrent genotyping. The mechanism used is substantially different than the arrayCGH mechanism in that the technique is not comparative. No reference sample is used and no co-hybridization is performed, and the method for copy number assignment relies on quantification of individual alleles and subsequent ratiometric analysis in contrast to arrayCGH where individual alleles are not assessed. Disadvantages of SNP arrays include increased noise levels, longer protocols, complexity of data interpretation and ethical implications, and possibly lower applicability to haploid samples.
The molecular cytogenetic technique of FISH (fluorescence in-situ hybridization), which uses chromosome-specific DNA probes, has frequently been applied to PGS and gives detectable signals on interphase nuclei. Although no amplification step is required, a significant disadvantage exists is that only a limited number of chromosomes can be assessed concurrently, limited by the number of distinct colors available for labeling of the DNA probes. The most comprehensive FISH methods used for routine embryo screening currently assess only half of the chromosomes, and thus, some chromosomal abnormalities are missed. Other disadvantages of FISH include overlapping signals which are difficult to score.